Abstract Heart failure affects six million people in the United States, and is listed as a causative factor in more than 10% of deaths. The development of heart failure is linked to several risk factors (including coronary artery disease, obesity and diabetes), which are increasingly prevalent in Western societies due to diet and other lifestyle choices. While clinical outcomes have improved over the last three decades, there remain gaps in our knowledge surrounding the cellular mechanisms that regulate cardiac function. One such gap, and the scientific focus of this application, is the regulation of fuel substrate utilization by mitochondria in the heart. Mitochondria provide 95% of the energy required by healthy hearts to maintain contractility, and defects in mitochondrial bioenergetic activity lead to cardiac energy starvation and heart failure. Mitochondria in the heart normally provide this energy through the oxidation of fatty acids; however, during heart failure they switch to other fuels like glucose. While changes in cardiac substrate preference in heart failure have been well characterized, we do not fully understand the cellular mechanisms that regulate this process. Our data, and the current literature, show that mitochondrial function is regulated by lysine acetylation, a post-translational modification that uses fuel-derived acetyl-CoA as a substrate. We recently identified GCN5L1 as the first component of the mitochondrial acetyltransferase machinery, and showed that GCN5L1-mediated acetylation controls mitochondrial bioenergetics in vitro. The objective of this proposal is to understand how GCN5L1 acetylation impacts mitochondrial bioenergetics in the heart, and to investigate how dysregulated energy substrate utilization can lead to mitochondrial dysfunction, cardiac energy depletion and heart failure. We will achieve this objective by addressing the following questions: (1) How does GCN5L1 control fatty acid oxidation in normal and failing hearts? (2) What acetyl modifications regulate mitochondrial fuel utilization enzymes during early- and late-stage heart failure? (3) How does GCN5L1 regulate cardiac mitochondrial turnover under normal and energy-depleted states? To answer these questions, we will use a series of in vivo murine heart failure models and in vitro cell culture studies, combined with metabolomic, proteomic and biochemical techniques, to examine the biology of GCN5L1. We expect that this series of experiments will provide important new insights on mitochondrial energy substrate regulation, and will highlight GCN5L1 as a crucial component in the control of metabolic fuel choice, bioenergetics and mitochondrial turnover in the heart.